4.»ùÒò×é·ÖÎö £¨Genome analysis\)

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1 4. 基因组分析 (Genome analysis) 4.1 Genome sequencing 4.2 A impossible mission: To sequence a genome completely 4.3 Gene finding

2 4.1 Genome sequencing From history of human genome project Strategic issues for sequencing large repeat-rich genomes Appearance of genomes

3 4.1.1 History of the Human Genome Project Watson, Crick DNA structure Berg, 1st recombinant DNA Botstein, Davis, Skolnick Maxam, Gilbert, Sanger sequence White DNA propose to map human genome with RFLPs Wada proposes to build automated sequencing robots MRC Sinsheimer publishes hosts first large meeting to genome discuss HGP Epstein-Barrat UCSanta virus (170 kb) Cruz; Kary Mullis develops PCR DOE begins genome studies with $5.3 million Gilbert announces plans to start company to sequence and copyright DNA; Burke, Olson, Carle develop YACs; Donis-Keller publish first map (403 markers)

4 History of the Human Genome Project (continued) 1987 (cont) Hood NIH produces supports the first HGP; automated Watson sequencer; heads the Dupont project and devolops allocates fluorescent part of the dideoxy- budget to nucleotides study social and ethical issues Hood, Olson, Botstein Cantor propose using STS s to map the human genome Proposal Venter to sequence announces 20 Mb in strategy to model sequence organism by ESTs. He 2005; plans to Lipman, Myers publish the BLAST algorithm patent partial cdnas; Uberbacher develops GRAIL, a gene finding program Simon develops BACs; US and French teams publish first physical maps of chromosome s; first genetic maps of mouse and human genome published Collins is named director of NCHGR; revise plan to complete seq of human genome by 2005 Venter publishes first sequence of free-living organism: H. influenzae (1.8 Mb); Brown publishes on DNA arrays Yeast genome is sequenced (S. cerevisiae)

5 History of the Human Genome Project (continued) Blattner, Plunket complete E. coli sequence; a capillary sequencing machine is introduced. SNP project is initiated; rice genome project is started; Venter creates new company called Celera and proposes to sequence HG within 3 years; C. elegans genome completed NIH proposes to sequence mouse genome in 3 years; first sequence of chromosome 22 is announced Celera and others publish Drosphila sequence (180 Mb); human chromosome 21 is completely sequenced; proposal to sequence puffer fish; Arabadopsis sequence is completed Celera publishes human sequence in Science; the HGP consortium publishes the human sequence in Nature

6 History of human genome project

11 The number of genomes sequenced each year since 1995

13 Citation tree for the WGS sequencing of H. influenzae

14 Citation tree for the WGS sequencing of E. coli

15 Genomes in numbers Sizes: virus: 10 3 to 10 5 nt bacteria: 10 5 to 10 7 nt yeast: 1.35 x 10 7 nt mammals: 10 8 to nt plants: to nt Gene number: virus: 3 to 100 bacteria: ~ 1000 yeast: ~ 7000 mammals: ~ Plants: ?

16 Sequencing projects «small» genomes (<10 7 ): bacteria, virus Many already sequenced (industry excluded) More than 90 microbial genomes already in the public domain More to come! (one new every two weeks ) «large» genomes ( ) eucaryotes 12 finished (S.cerevisiae, S. Pombe, E. cuniculi, C.elegans, D.melanogaster, A. gambiae, D. rerio, F. rubripes, A.thaliana, O. sativa, M. musculus, Homo sapiens) Many more to come: rat, pig, cow, maize (and other plants), insects, fishes, many pathogenic parasites (Plasmodium ) EST sequencing Partial mrna sequences ~12x10 6 sequences in the public domain

17 基因组地理探险 : 四张图哥伦布几百年前发现了美洲澳洲等新大陆, 最终使人类认识了地球上的每一块处女地于是有人形象地把人类目前的基因组研究形象地比喻为地球探险, 并把基因组研究称为基因组地理 (genomic geography) 我们当前的测序技术还不允许我们把整条染色体拿来测序, 而只能一小段一小段 ( 一般一个测序反应为 500 个碱基对长度 ) 地进行这些小段长度 (500bp) 的 DNA 片段对于整条染色体而言, 就仿佛是染色体长线上的一个个小点, 那么如何把这些测序完成的小片段连接在一起而获得整条染色体的序列呢? 这是人类基因组计划的最大难题之一

18 A 遗传图谱遗传学图距是以形成精子或卵子的减数分裂过程中, 两个位点之间进行交换重组百分率 (cm, 厘摩尔根 ) 为单位的, 反映基因遗传效应的基因图谱限制性片段长度多态性 (RFLP) \ 微卫星标记 (microsatellite marker) \SNP (single nucleotide polymorphysm) 的遗传标记系统, 即单核甘酸多态性

19 B 物理图谱物理图以 Mb kb bp 作为图距, 以 DNA 探针的 STS(sequence-tagged site, 序列标签位点 ) 序列为路标构建物理图谱的一个主要内容是把含有 STS 对应序列的 DNA 的克隆片段连接成相互重叠的片段重叠群 (conting) 在 YAC 载体的基础上, 现有 BAC( 细菌人工染色体库 ) PI( 一种源于 λ 噬菌体的载体 ) PAC( 来源于 PI 的人工染色体 ) MAC( 一种类似于 YAC, 但以哺乳细胞作为宿主细胞的新型载体 ) Fosmid( 一种类似于 BAC, 来源于大肠杆菌的 F 基因的载体 )

20 C 序列图谱由全部核甘酸组成的基因组序列图前面所谈的遗传图与物理图的构建都是为了绘制序列图而建的 1977 年, 人类完成对自然界第一个基因组 ( 全长 5.3kb 的 φx174 噬菌体 ) 全序列测序, 整个测序历时将近一年时间 1987 年第一台自动测序仪投入使用后,1990 年完成了长 230kb 的人巨细胞病毒 (HCMC) 的全序列测序工作后来又得到了第一个细菌基因组全序列 (1995, 1.9Mb) 模式生物酶母基因组全序列 (1996, 12Mb) 线虫基因组全序列 (1998,97Mb) 果蝇基因组全序列 (1999,136Mb), 直到人类自身全序列 (2000,3286Mb) 水稻 (2001,450M)

21 D 基因图谱基因图谱就是基因组中全部基因的位置结构与功能的明细图基因图谱的意义在于它能有效地反映在正常或受控条件中表达的全基因的时空图

22 4.1.2 基因组测序的策略 DNA 测序的一般方法 : 进行 DNA 序列测定时, 在 4 个独立的反应中, 各产生一套放射性标记的单链寡核苷酸, 它们有固定的起点, 另一端终止于不同的 A T G 或 C 位点每个反应的产物在高分离度的聚丙烯酰胺凝胶上电泳分级经放射自显影, DNA 序列可从凝胶上直接读出 ( 奥斯伯等,1998) 双脱氧测定法 (Sanger 法 ) 化学测序法 (Maxam-Gilbert 法 )

23 荧光自动测序仪 4 种碱基产生 4 种颜色的荧光反应, 以单泳道或毛细管电泳就可以分辨出相应的寡核苷酸产物

24 两个基因组测序策略 : 逐步克隆方法 (clone by clone) 全基因组鸟枪方法 (whole genome shotgun,wgs)

25 Clone by Clone Approach

26 The key steps in assembling individual sequenced clones into the draft genome sequence

27 Assembling the Draft Sequence of indica Rice Assembler: RePS (Repeat-masked Phrap with Scaffolding)

29 Quality: 546 bp at Q20 (indica rice genome) Contigs:127,550 (N50=6,688 bp) Scaffolds: 102,444 (N50=11,764 bp)

30 4.1.3 Appearance of genomes One to many chromosomes Repeat sequences common in some genomes e.g. 35% of human are transposable elements -10% Alu, 14.6% LINE1 sequences Gene structure varies no. and length of introns What does 50 kb of sequence look like? repeat Pseudogene Intron-exon components of a gene Human very few genes - repeats Yeast many genes (~25) few repeats Maize mostly repeats

31 Human Genome

32 The genome of T. tengcongensis

33 The genome of T. tengcongensis

34 MDRs in human genome MDRs in rice genome

35 The Reverse-phase of Gene-jungles : Repeat-jungles, But No Deserts! MDRs in rice genome MDRs in human genome

36 1 exact 20-mer repeats in O. sativa fraction of data cdna (exon) intron transposon log10 (copy number)

37 fraction of data exact 20-mer repeats in O. sativa cdna (exon) intron transposon log10 (copy number) Plant Repeats Are Mostly Not Found In Introns But In Intergenic Regions. 1 exact 20-mer repeats in A. thaliana 1 exact 20-mer repeats in H. sapiens fraction of data cdna (exon) intron transposon log10 (copy number) fraction of data cdna (exon) intron transposon log10 (copy number)

38 Jungle-Desert Or Big Small? The Gene Jungle The Gene Desert Gene Space ~50% Intergenic Intergenic Intergenic Many Small Genes Two Big Genes Gene Space ~90% Intergenic Intronic Intergenic Intronic

39 Bimodal distribution of introns: Spikes and Humps Human Frog Mouse Fruitfly Chicken Nematode

40 Exon-Intron Organization In Plant Genomes Arabidopsis resembles nematode. Rice resembles cold-blooded vertebrates.

41 GC-Content Comparison of Exons and Introns Exon GC Human Genomic Length, Kb Intron GC Human Genomic Length, Kb Exon GC Mouse Genomic Length, Kb Intron GC Mouse Genomic Length, Kb

42 A Major GC-content Increase in Rice

43 Not Between-gene, But Within-gene Dicot Plant Intron GC Intron GC 0.5 Monocot Plant Genomic Length, Kb Genomic Length, Kb

44 No-hit Genes Are Real Genes. ~15% EST+ ~45% EST+

45 Identity Between Indica Cultivars Differences Are almost All in Repeats

46 4.2 A impossible mission: To sequence a genome completely 有没有可能将基因组测序完成? 随机克隆对基因组的覆盖问题

47 随机克隆对基因组的覆盖问题 : 问题 : 基因组长度为 G, 随机切成长度为 L 片段 ( 克隆增殖,L=15kb),G L, 取 N 个克隆来, 问对原来基因组覆盖率多少? 在均匀分布的假设下, 一定碱基在指定的克隆中的概率为 L/G; 不在的概率为 1-L/G 不在所有 N 个克隆中的概率为 (1-L/G) N =(1-(LN/G) (1/N)) N e -LN/G LN=XG, (X=LN/G, coverage 覆盖率 ), 则上式为 e -x 则一定碱基在 N 个克隆中的概率为 f=1- e -x (Clarke-Carbon, 1976)

48 X f Poisson 分布 :P(y)=(t y e t ) /y!, P(0)=e t t=x

49 Lander-Waterman 曲线 Lander ES, Waterman MS. Genomic mapping by fingerprinting random clones: a mathematical analysis. Genomics Apr;2(3): 平均 contig 数 (XExp(-X)) 理论曲线实际曲线覆盖率 (X)

一步一步教你使用NCBI

一步一步教你使用NCBI 查找 DNA mrna cdna Protein promoter 引物设计 BLAST 序列比对等作者 :urbest 2007-8-1 苏州大学生命科学学院最近看到很多战友在论坛上询问如何查询基因序列如何进行引物设计如何使用 BLAST 进行序列比对, 这些问题在 NCBI